Isolation of dominant gain-of-function alleles of EXO84. (A) Single extra-chromosomal (CEN) copies of wild-type SRO7, wild-type EXO70, a gain-of-function allele EXO70-I114F, and wild-type EXO84 or multicopy (2μ) copies of wild-type SRO7 and SRO7-A5,A6 were transformed into the sec2-41 temperature-sensitive mutant. The growth of three independent transformants is shown for each transformation under permissive and restrictive conditions. (B) Schematic for the gap repair screen to generate and identify dominant EXO84 gain-of-function alleles in the sec2-41 mutant. (C) The N12I, L365W, and E626V gain-of-function alleles of EXO84 were transformed into the sec2-41 mutant. The growth of three independent transformants is shown for each condition. (D) Wild-type EXO84 and two gain-of-function mutations EXO84-L365W and EXO84-E626V were integrated into the EXO84 chromosomal locus marked with nourseothricin (Nat) resistance and crossed to a sec2-41 strain. Following tetrad analysis (Fig. S2), six representative segregants containing both sec2-41 and indicated genotypes of EXO84 or EXO84 gain-of-function alleles were examined for their ability to suppress the temperature-sensitive growth of sec2-41 at the indicated temperatures. (E) The structure of S. cerevisiae Exo84 (residues 169–753; PDB accession no. 5YFP, chain H) with the domains annotated and gain-of-function residues N12, L365, and E626 indicated as yellow space-filled spheres.